1. Field of the Invention
This invention is directed to methods for the production of useful steroids and steroidal glycosides, including radio-labeled steroids and steroidal glycosides, by the establishment and maintenance of serially propagated cultures of Yucca plants, i.e., cell, root-cell and shoot cultures, and extraction of steroids and steroidal glycosides from the cultures. The plant tissue cultures are a unique biological source of Yucca steroidal glycosides (saponins).
2. The Prior Art
The initiation, maintenance and uses of various plant tissue culture systems have been well documented (in Plant Tissue and Cell Culture, Botanical Monographs, Volume 11, H.E. Street (Ed), University of California Press, 1977). Stohs, Rosenberg and Billets, Planta Medica 27, 257-261, 1975, have published a comprehensive review of the literature on steroid and sterol metabolism in plant cultures.
Various Yucca plant species produce steroidal saponins (Rothman et al, U.S. Pat. No. 2,715,122; Wall et al, U.S. Pat. No. 2,895,953). Sapogenins have been isolated from various parts of the Yucca plant (Stohs and Rosenberg, Lloydia 38(3), 181-194, 1975). The major steroids of Yucca are sarsasapogenin, its isomer smilagenin, markogenin, tigogenin, hecogenin, markogenin and gitogenin.
The use of diosgenin as a precursor of steroid hormones led to the investigation of plant tissue cultures as a source of steroids. Jhang, Staba and Kim (In Vitro 9(4), 253-259, 1974) established undifferentiated callus and cell suspensions of Panax quinquefolium (American Ginseng) and Panax ginseng (Korean Ginseng) which produced approximately 4% dry weight of steroidal glycosides. Metz & Lang received German Pat. No. 1,216,009 (1966) and Furuya received Japanese Pat. No. 48-31917 (1973) for Ginseng saponin production by ginseng root or cell cultures. There are several reports on the production of the steroid diosgenin by undifferentiated tissue cultures of Dioscorea species (Marshall and Staba, Phytochemistry 15, 53-55, 1976; Tai and Goldberg, Planta Medica 44, 107-110, 1982; Tomita et al, Phytochemistry 9, 111-114, 1970). Stohs et al (Phytochemistry 13, 2145-2148, 1974) incorporated [4-.sup.14 C-22, 23-.sup.3 H] sitosterol into diosgenin of Dioscorea deltoidea cell suspensions. Stohs et al (1974, supra) indicated that undifferentiated callus cultures of Yucca glauca grown on solid medium produced the sapogenin gitogenin and trace levels of the sapogenin markogenin. However, when the callus culture was passed into liquid medium and grown as a suspension culture, the ability to biosynthesize steroidal sapogenins was limited (Stohs et al, 1975, supra). Quintero et al (IAPTC Abstr., 1974, July 11-16, 1982, Tokyo) reported Yucca filifera callus cells to contain the steroid sarsasapogenin. Jain, Rosenberg and Stohs (Planta Med. 31: 109-111, 1977) isolated the sapogenins tigogenin and gitogenin from callus cultures of Trigonella foenium-graecum.
To date, the production of Yucca steroidal glycosides from Yucca tissue cultures has not been reported.